New approaches will be developed to examine the dynamics of signaling protein activation in living cells. These methods will be based upon novel, environmentally-sensitive fluorescent dyes designed to respond to protein conformational changes in vivo. Generally applicable methods and dyes will be developed by designing biosensors for the nucleotide state of Cdc42, Rac, and Rho. The new approaches will provide the ability to study endogenous, unlabeled proteins in living cells, to study activation of multiple proteins in the same cell, and to examine proteins buried within multiprotein complexes. The dyes will be very bright and undergo large fluorescence changes, enabling us to use low biosensor concentrations for minimum perturbation of normal cell physiology. The dyes can be used to obtain many images before photobleaching, providing excellent temporal resolution, and quantitation of changes in protein activation level over time in individual cells. Biosensors of Rac and Cdc42 will be imaged in the same cell, to examine how Cdc42 activation of Rac coordinates the activity of the two GTPases for motility. Finally, we will develop means to convert antibody fragments into biosensors, providing access to many protein activities. The new dyes will make it possible to use highly reversible antibody binding, to minimally perturb cell behavior. A monoclonal antibody specific for phosphorylated alpha4 integrin will be used as a model for development of the approach, and to study alpha4 regulation of Rac in directed cell movement.